The subject invention is directed at an oil-in-water emulsion in which recombinant collagen-like polymer is applied as a stabiliser. The stabilising effect occurs already at the stage of formation i.e. on the initial size of the droplets in the emulsion. Also the stabilising effect is visible when assessing the ageing of the oil-in-water emulsion. In both cases the droplet size is significantly reduced vis-à-vis the prior art oil-in-water emulsions comprising gelatin. The stabilising effect occurs at a range of temperatures and a range of pH values. It now in fact has become possible to operate processes requiring oil-in-water emulsions at lower temperatures than was possible to date and also at lower pH values than normally are applied to date. The same holds true for the storage temperature and pH at which the oil-in-water emulsions according to the invention can currently be maintained. The oil-in-water emulsions can now be stored longer than was previously the case. Also the oil-in-water emulsions according to the invention can be as stable as the prior art oil-in-water emulsions comprising gelatin at lower concentrations of surfactant than used in the prior art oil-in-water emulsions.
In addition the subject invention provides the possibility to use recombinant collagen-like polymer which are composed of polar and apolar end tails for oil-in-water emulsions and also provides for the first time the bipolar, or more specifically amphiphilic, compounds as such and a description of a method to achieve the production of such compounds.
Oil-in-water emulsions consist of hydrophobic droplets in a hydrophilic continuous phase. The interfacial area between these hydrophobic droplets and the hydrophilic continuous phase is stabilised with surfactants and/or polymers.
In the manufacturing process, the size of the droplets in the oil-in-water emulsion is a factor needing careful control. The average size of the droplets in the oil-in-water emulsion should be small i.e. the initial size of the droplets should be as low as possible. Also the size stability of the droplets after making the oil-in-water emulsion should be high i.e. the ageing stability should be as high as possible thus ensuring the increase in droplet size in time is kept as low as possible. To realise this small initial size and this limited ageing, the oil droplets can be stabilised by gelatin.
Present stabilisation methods however have several disadvantages:
1. The initial size of the oil-in-water emulsion is rather large.
2. The stabilisation capability of the present gelatin is limited, meaning that in the manufacturing process the oil-in-water emulsions have a limited life time in which they can be applied for specific functions.
The presently used polymer-like materials (like gelatin) originate from natural sources and the structure and the related rheological and surface chemical characteristics can be modified only in a limited manner, J. Colloid Polym. Sci. 272: 433-439 for example reveals experimental data about the relation between the molecular mass distribution of non-recombinant natural gelatin and it""s effectiveness in the stabilisation of oil-in-water emulsions. In the case of gelatin samples with a content of more than 30% of the low molecular weight fraction as described in the article an improved stabilisation was obtained in comparison to the native non recombinant non hydrolysed gelatin. The problem still remained with this modified gelatin that the reproducibility of such processes using natural gelatin sources is not extremely good. In particular this is a preferred requirement for photographic applications.
In addition when considering use of oil-in-water emulsions for consumption purposes e.g. in foodstuffs the risk associated with mad cows disease for example can have a prohibitive effect on the use of gelatin derived from natural sources as a stabiliser.
The invention is directed at an oil-in-water emulsion comprising recombinant collagen-like (or gelatin-like) polymer in an amount sufficient to act as a stabiliser of the emulsion. The advantages thereof are described in detail elsewhere in the description. An oil-in-water emulsion according to the invention suitably is one wherein the recombinant collagen-like polymer is free of triple helix structure. The recombinant collagen-like polymer is suitably free of any helix structure. It is a preferred embodiment of the invention that the recombinant collagen-like polymer of the oil-in-water emulsion is free of hydroxyproline as this ensures the absence of (triple) helix formation. The triple helical structure is present in natural gelatin. The absence of the (triple) helical structure is advantageous, because the emulsification can be operated at lower temperatures (15-40xc2x0 C.) than the traditional temperature during the emulsification process (T higher than 40xc2x0 C.).
The method of arriving at recombinant collagen-like polymer has been described in detail in commonly owned U.S. Pat. No. 6,150,081, inventors van Heerde et al., for example at column 14, line 48 to column 15, line 17, at column 22, line 51 to column 25 line 18 and elsewhere throughout the specification, the entire disclosure of which patent is hereby incorporated herein by reference thereto. The methodology is described in the publication xe2x80x98High yield secretion of recombinant gelatins by Pichia pastorisxe2x80x99, M. W. T. Werten et al., Yeast 15, 1087-1096 (1999), in press.
To be defined as collagen-like at least one GXY domain should be present of at least a length of 5 consecutive GXY triplets and at least 20% of the amino acids of the recombinant collagen-like polymer should be present in the form of consecutive GXY triplets, wherein a GXY triplet consists of G representing glycine and X and Y representing any amino acid. Suitably at least 5% of X and/or Y can represent proline and in particular at least 5%, more in particular between 10 and 33% of the amino acids of the GXY part of the recombinant collagen-like polymer is proline. For the purposes of this patent application the recombinant collagen-like polymer consists of at least 4 different amino acids, preferably more than 10 different amino acids, more preferably more than 15 different amino acids. It can comprise any of the amino acids known. A preferred oil-in-water emulsion according to the invention is one, wherein the recombinant collagen-like polymer comprises at least one lysine residue.
Any of the embodiments disclosed in the van Heerde et al. U.S. Pat. No. 6,150,081 can be applied for the oil-in-water emulsions according to the invention. A preferred embodiment of an oil-in-water emulsion according to the invention is one wherein the recombinant collagen-like polymer has an isoelectric point at least 0,5 pH units removed from the pH of the oil-in-water emulsion itself. Suitably one pH unit removed or even more. The advantage hereof is that the pH at which the emulsion needs to be maintained or used or prepared can vary depending on the isoelectric point (pI) of the applied recombinant collagen-like polymer. The recombinant technology enables variation previously unavailable for tailoring the polymer and thus tailoring the pI. It will be appreciated that not all processes requiring an oil-in-water emulsion are best carried out at pH 6 which is the pH value at which prior art gelatin comprising oil-in-water emulsions were optimally used. Naturally the pH=6 can also be used in those cases where it is still useful or in fact optimal. However the oil-in-water emulsions according to the invention no longer need the strict control of the pH during any of the processes e.g. preparation, storage or application as was previously the case. Now it has in addition become possible to use the oil-in-water emulsions according to the invention at pH=5. It has now become possible to develop oil-in-water emulsions with recombinant collagen-like polymers of extremely divergent pI values. Suitable embodiments involve pI anywhere from 4-10. pI equal to or higher than 6, equal to or higher than 7 and even equal to or higher than 8 and higher than 9 have been achieved and they are illustrated in the examples. We also illustrate pI selected from 4-7. The presence of collagen-like polymers with an isoelectric point far from the actual pH of the OW (oil-in-water) emulsion according to the invention is preferred. Such a pH has the advantage that the overall charge and the overall three dimensional conformation of the polymer is independent of the pH, and so the steric stabilisation of the OW emulsion is also independent of the pH.
An oil-in-water emulsion according to the invention will use recombinant collagen-like polymer with a molecular weight of at least 2.5 kDa. Suitably the molecular weight is lower than 170 kDa, preferably lower than 100 kDa. We have found improved results when the molecular weight is higher than 20 kDa, preferably higher than 25 kDa and even more preferably higher than 50 kDa. A preferred range thus goes from 20 kDa to 100 kDa.
An oil-in-water emulsion according to the invention, which is particularly useful, is one, wherein the recombinant collagen-like polymer is present in a hornodisperse size distribution. A homodisperse size distribution means that the optimal size distribution and the uniformity and reproducibility can be guaranteed for the desired application. According to the invention, the notion xe2x80x9chomodispersexe2x80x9d preferably means that at least 75% of the molecules have a molecular weight between xe2x88x9210% and +10% of the average molecular weight. It is clear for example that steric hindrance is limited in cases where the size is too low. A size that is too high causes a high viscosity, which is inconvenient for the emulsion equipment and for the emulsification process. The invention now provides the opportunity to regulate this in an optimal manner. A lower viscosity enables application of higher concentrations of the gelatin and the oil. Thus the ratio of oil versus water can be improved, which will be advantageous for several photographic applications.
In an alternative embodiment an oil-in-water emulsion according to the invention, is one wherein the recombinant collagen-like polymer is present together with non recombinant collagen i.e. an oil-in-water emulsion which comprises a mix with natural gelatin or prior art gelatins can also be used. Surprisingly good results concerning stability vis-à-vis initial size and ageing stability are possible. No phase separation occurs and dissolving occurs only in the water, which is particularly interesting for example in photographic application. In a suitable embodiment the oil-in-water emulsion according to the invention can be one, wherein the recombinant collagen-like polymer is present together with non recombinant collagen in a ratio of 99%-20% on weight basis of recombinant collagen-like polymer on the basis of total weight of collagen-like polymer in the oil-in-water emulsion. The initial size of the oil-in-water emulsion resulting from this mixing process, stabilised by said protein-like material made by genetic engineering, was smaller than the initial size of the oil-in-water stabilised by traditional polymer-like material, and the ageing characteristics of said oil-in-water emulsion were improved, under a wide variety of conditions (variation in T, surfactant, pH, polymer-like stabiliser combinations, etc.), as compared with the prior art.
Of particular interest is the fact that oil-in-water emulsions according to any of the embodiments of the invention exhibit better initial size characteristics as can be determined by measuring the droplet size at a particular pH and temperature of the emulsion and measuring the size under the same conditions for a prior art oil-in-water emulsion. A suitable test revealing better initial size characteristics can comprise measuring a smaller initial droplet size at T=40xc2x0 C. or less and pH=5 at 2 ml scale using ultrasonic technique in comparison to prior art gelatin under corresponding conditions, e.g. at a temperature T selected from the range of 10-40xc2x0 C., suitably 15-40xc2x0 C. e.g. T=30, 25, 20, 15 or 10xc2x0 C., wherein the comparison is optionally carried out in the presence of surfactant, e.g. in an amount corresponding to 0.4868 mM SDBS/5 grams of collagen-like polymer/liter. An improvement can comprise the oil-in-water emulsion according to the invention exhibiting better initial size characteristics as can be determined by measuring a smaller initial droplet size than 600 nm, preferably below 500 nm, even lower than 350 nm, 250 nm and more preferably below 200 nm at T=40xc2x0 C. or less e.g. at a T selected from the range of 10-40, suitably 15-40xc2x0 C. e.g. T=30, 25, 20, 15 or 10xc2x0 C. at pH=5, wherein the comparison is carried out optionally in the presence of a surfactant, e.g. in an amount corresponding to 0.4868 mM SDBS/5 grams of collagen-like polymer per liter.
Not only is an improvement of initial size often found, but also better ageing characteristics as can be determined by measuring the droplet size after a period of time at a particular pH and temperature of the emulsion and measuring the size under the same conditions for a prior art oil-in-water emulsion. An example of a suitable test to reveal this characteristic is by measuring an increase in droplet size after 4 hours at T=40xc2x0 C. or less and pH=6 at 2 ml scale using ultrasonic technique in comparison to prior art gelatin under corresponding conditions, e.g. a T from the range 10-40, suitably 15-40xc2x0 C. e.g. 1-30, 25, 20, 15 or 10xc2x0 C., wherein the comparison is optionally carried out in the presence of surfactant, e.g. in an amount corresponding to 0.4868 mM SDBS/5 grams of collagen-like polymer/liter. Suitably one will find for oil-in-water emulsions according to the invention better ageing characteristics as can be determined by measuring a smaller increase in droplet size after 4 hours than 450 nm, preferably below 400 nm, preferably below 350 nm and more preferably below 300 nm and even below 250 nm at T=40xc2x0 C. or less e.g. at a T selected from the range of 10-40xc2x0 C., suitably 15-40xc2x0 C. e.g. T30, 25, 20, 15 or 10xc2x0 C. at pH=6, wherein the comparison is carried out optionally in the presence of surfactant, e.g. in an amount corresponding to 0.4868 mM SDBS/5 grams of collagen like polymer/liter.
The tests can be carried out at different pH values depending on the recombinant collagen-like polymer used in the oil-in-water emulsion. The improvement is generally more noticeable at lower temperatures and at pH lower than those generally used for prior art gelatins i.e. at a pH lower than 6 suitably lower than 5.5 e.g. around 5 or lower.
An oil-in-water emulsion according to any of the embodiments of the invention will not exhibit gelation at a temperature below 30xc2x0 C.
Oil-in-water emulsions according to any of the embodiments of the invention will exhibit increased stability in the presence of surfactant at a concentration below that equivalent to 1 mmol SDBS/5 gram gelatin/l as can be determined by measurement of droplet size increase after 4 hours at pH 6.0 and T=40xc2x0 C. below 250 nm.
An oil-in-water emulsion according to any of the embodiments of the invention can comprise the recombinant collagen-like polymer in concentrations of collagen-like polymer in the range of 2-100 gram/l solvent, in particular between 5 and 50 g/l solvent. This is advantageous in comparison to the prior art oil-in-water emulsions i.e. higher gelatin concentrations are feasible than oil-in-water emulsions using gelatin applied in the prior art.
An oil-in-water emulsion according to any of the embodiments of the invention can exhibit a viscosity in the range 0.005-8 mPa when dissolved in a concentration of 6.6% in water at a temperature of 40xc2x0 C.
Due to the development of the recombinant technology it has now become possible to develop for use specifically in oil-in-water emulsion according to any of the embodiments of the emulsions according to the invention recombinant collagen-like polymer exhibiting an amphiphilic structure, with one end of the molecule being polar and the other end being apolar e.g. wherein the recombinant collagen-like polymer exhibits an amphiphilic structure, with one end of the molecule being polar due to the presence of a sufficient number of polar amino acid residues to render that end polar and the other end being apolar due to the presence of a sufficient number of apolar amino acid residues to render that end apolar. Collagen-like polymers with an amphipolar character (one side hydrophilic, one side hydrophobic) show an optimal interfacial behaviour and have a strong preference for a position on the oil-water interface (with one leg in the oil-phase and xe2x80x9cone legxe2x80x9d in the water-phase, resulting in a low interfacial tension) by which the initial size and stabilisation are optimised. The manufacture of the polar hydrophilic collagen molecule can be made following the detailed method described in van Heerde et al. U.S. Pat. No. 6,150,081. Obviously, the changes required in the amino acid sequence can be achieved in a manner well known to the skilled person when wishing to introduce a few specific amino acid substitutions. The skilled person also knows which amino acids can be substituted and which amino acids can be used to enhance polarity or apolarity. The polar and apolar constructs can be combined using standard methodologies of ligation for the manufacture of the bifunctional collagen-like polymer. Not only is an oil-in-water emulsion as such part of the invention but also any of the bipolar molecules as such and a process for making them. An amphiphilic recombinant collagen-like polymer i.e. polar at one end and apolar at the other to a degree sufficient for the polar end to extend into a water phase and the apolar end to extend into an oil phase, wherein recombinant collagen-like is further as described for any of the recombinant collagen-like polymers as components of an oil-in-water emulsion according to the invention, is thus also covered.
The amphiphilic nature of the preferred collagen-like polymers of the invention can be defined with reference to the transfer free energy of the individual amino acids constituting the polar and apolar parts of the polymer, respectively. This transfer free energy (xcex94F) is the energy (in kcal/mole) of the amino acid residue in an xcex1-helix to be transferred from the membrane interior to the water phase. These energy values as defined by Engelman et al, Ann. Rev. Biophys. Biophys. Chem. 15 (1986), 330, are summarised in the table below.
The polarity of a given amino acid sequence is defined herein as the average transfer free energy per amino acid of the sequence, which equals the sum of the product of the number of individual amino acids and the transfer free energy of each amino acid, divided by the total number of amino acids. In a formula:
Polarity=(xcexa3nixcex94Fi)/nt
wherein ni is the number of each individual amino acid, xcex94Fi is the transfer free energy of the corresponding amino acid, and nt is the total number of amino acids. As an example, a 15-mer apolar peptide having the following amino acid sequence:
Gly Pro Pro Gly Val Pro Gly Phe Ile Gly Phe Pro Gly Leu Pro has the following amino acid composition: 5 Gly+5 Pro+1 Val+2 Phe+1 Ile+1 Leu, and hence it has the following polarity (in kcal/mole per amino acid):
(5*1.0+5*xe2x88x920.2+1*2.6+2*3.7+1*3.1+1*2.8)/15=19.9/15=+1.33
Apolar sequences generally have positive polarity values, whereas polar sequences have negative polarity values. According to the invention, amphiphilic collagen-like polymers have a polar part and an apolar part, the polar part having a polarity value which is at least 0.3 lower (i.e. less positive or more negative), preferably at least 0.5 lower, more preferably at least 0.7 lower than the apolar part. The polar part an the apolar part may be separated by a bridge, the polarity of which may be intermediate. It is preferred that the polar part and apolar part each make up at least 10% of the total length (defined in chain atom numbers) of the polymer, preferably each at least 20% of the length. In particular each part (polar and apolar) contains at least 10, more in particular at least 20, most particularly at least 30 amino acids, up to half of the total number of amino acids. Preferably the polar and apolar parts are located at the two opposite ends of the polymer, with preferably less than 5%, or less than 10 amino acids, and most desirably no amino acids being located at the outer ends beyond the polar and apolar parts.
In a preferred embodiment, the polar part of the amphiphilic polymer contains at least 10% (on the basis of the number of amino acids), preferably at least 15%, of polar amino acids selected from Arg, Asp, Lys, Glu, Asn, Gln and His, whereas the apolar part contains at least 10%, preferably at least 15%, of apolar amino acids selected from Phe, Met, Ile, Leu, Val, Trp and Ala. In both parts, at least about 15, preferably at least 30% will be Gly, and at least 10% will be Pro. Preferably, the polar part contains less than 10% (more preferably less than 7%) of the apolar amino acids selected from Phe, Met, Ile Leu, Val, Trp and Ala and the apolar part contains less than 10% (more preferably less than 7%) of the polar amino acids selected from Arg, Asp, Lys, Glu, Asn, Gln and His.
The amphiphilic polymer may also comprise alternating polar and apolar stretches, each stretch being e.g. between 5 and 100, preferably between 10 and 50 amino acids in length. The number of alternating stretches may be two up to e.g. ten of such stretches (pairs of polar and apolar stretches). At least one polar stretch of such series, preferably two or more stretches, more preferably at least the terminal polar stretch, and most preferably each polar stretch has a polarity difference with the apolar stretch or preferably the apolar stretches as defined above. In this alternating arrangement, each pair of polar and apolar stretches may be separated from the next pair by an indifferent bridge of intermediate polarity.
Obviously an oil-in-water emulsion according to any of the embodiments of the invention described above as such or any combination thereof is covered by the invention. Also any such oil-in-water emulsion can comprise further additives rendering it particularly suited to the application purpose of the emulsion. By way of example for the preferred application in photography, said additive can be selected from any of the following group of components, said group consisting of coupler, dye, organic solvent, inorganic solvent, surface/interface active agent, scavenger, UV absorber, optical brightener, stabiliser, pH controlling agent, mono/divalent ions. In the case of application in foodstuffs, pharmaceuticals or cosmetics the additives must be non toxic i.e. pharmacologically acceptable to humans and/or animals.
Examples of protein-like structures, which can be applied for stabilisation of OW emulsions, are provided in the experimental description elsewhere. In the examples the improvement of the oil-in-water emulsion stability and the oil-in-water emulsion initial size is illustrated by use of various recombinant collagen-like polymers under various conditions. For example homodisperse molecules of varying sizes have been used. Molecules in which helical structure is absent have been used. Molecules with an pI of 9 have been used. The pH dependence and the T dependence of the OW emulsion stability and initial size are shown.
Oil-in-water emulsions are made by mixing a solution of collagen-like material in the hydrophilic phase with a hydrophobic phase. Mixing can be executed by stirring, by high-pressure homogenisation, by treatment with ultrasonic frequencies, or the like. The hydrophobic phase can be any hydrophobic liquid suitable for the intended use. For example, trialkyl phosphates and triaryl phosphates such as trihexyl, trioctyl, tridecyl, tris(butoxyethyl), tris(haloalkyl), trixylenyl and tricresyl phosphate, can be used for preparing photographic emulsions. Also phthalate esters, citric esters, benzoic esters, fatty acid esters and fatty acid amides, as well as hydrocarbons such as n-decane or n-dodecane can be used. Edible triglycerides derived from vegetable or animal fats can be used for preparing emulsions for use in nutritional, cosmetic and pharmaceutical products, etc. Surfactants, such as sodium dodecylbenzenesulphonate, can be added and oil-soluble components such as precursor molecules for dyes and UV absorbers, and further reducing reducing agents and other compounds can be added. Temperature can be varied. The protein-like material can consist of a pure component homodisperse) or a mixture of components, all made by genetic engineering, or it can consist of a mixture of a component made genetic engineering and a traditional polymer. The invention covers a process comprising application of an oil-in-water emulsion according to any of the embodiments provided as oil-in-water emulsions according to the invention. Specifically the process can be a photography process or a foodstuff production process. Suitably a process according to the invention can be carried out at least at some stage in the presence of the oil-in-water emulsion at a pH below 6.0 preferably below 5.5 and suitably between 4.5-5.5. A process according to the invention can be carried out at some stage in the presence of the oil-in-water emulsion at a temperature below 40xc2x0 C., suitably at ambient temperature i.e. between 10-30xc2x0 C., suitably between 18-25xc2x0 C. i.e. in absence of a heating step, preferably during the whole process. A process comprising a combination of any of the steps mentioned falls within process protection claimed. A process comprising any of the above mentioned measures, said process being storage of an oil-in-water emulsion according to any of the embodiments of the invention is also covered by the invention as is a process of preparation of any of the embodiments of the oil-in-water emulsion according to the invention.
General remarks about advantages of the application of the recombinant collagen-like polymers over traditional gelatins:
Monodisperse products, creating the flexibility to design an OW emulsion with an optimal MW mix for creating steric hindrance without xe2x80x9cbridge makingxe2x80x9d coagulation behaviour.
Prevention of gelation bebaviour (when indicated), creating freedom of processing temperature.
Freedom to choose the isoelectric point and surface active behaviour (by polar/a-polar AA), which is for stabilisation and for robustness of stability in case of emulsion pH variations (=amphipolar collagen-like polymers).
Freedom to use lower surfactant concentrations to obtain comparable or even improved stability.
The recombinant collagen like polypeptide as defined above can be produced by expression of a collagen-like polypeptide encoding nucleic acid sequence by a suitable microorganism. The process can suitably be carried out with a fungal cell or a yeast cell. Suitably the host cell is selected from the group consisting of high expression host cells like Hansenula, Trichoderma; Aspergillus, Penicillium, Neurospora and Pichia. Fungal and yeast cells are preferred to bacteria as they are less susceptible to improper expression of repetitive sequences. Most preferably the host will not have a high level of proteases that attack the collagen structure expressed. In this respect Pichia offers an example of a very suitable expression system. Preferably the micro-organism is free of active post-translational processing mechanism for processing collagen like sequences to fibrils thereby ensuring absence of helix structure in the expression product. Also such a process can occur when the microorganism is free of active post-translational processing mechanism for processing collagen like sequences to triple helices and/or when the nucleic acid sequence to be expressed is free of procollagen and telopeptide encoding sequences. The host to be used does not require the presence of a gene for expression of prolyl-4-hydroxylase the enzyme required for collagen triple helix assembly contrary to previous suggestions in the art concerning collagen production. The selection of a suitable host cell from known industrial enzyme producing fungal host cells specifically yeast cells on the basis of the required parameters described herein rendering the host cell suitable for expression of recombinant collagen according to the invention suitable for photographic applications in combination with knowledge regarding the host cells and the sequence to be expressed will be possible by a person skilled in the art.
Several strong and tightly-regulated inducible promoters are available for yeast systems and other recombinant production systems, allowing a highly efficient expression and minimising possible negative effects on the viability and growth of the host cells. When, for example, the methylotrophic yeast Pichia pastoris is used, the integrative can be incorporated into the yeast""s genome after transformation of the host, resulting in genetical stability of the transformants (loss of plasmids is then of no importance). It is possible to generate transformants with the heterologous target gene under the control of e.g. the alcohol oxidase (AOX) promotor), in which the recombinant gene is either incorporated into the HIS4 locus or the AOX1 locus.
To ensure production of a non cleaved sequence a process according to the invention for producing recombinant collagen like material comprises use of a nucleic acid sequence encoding recombinant collagen amino acid sequence substantially free of protease cleavage sites of protease active in the expression host cell. In the case of Pichia pastoris for example and possibly also for other host cells a nucleic acid sequence encoding collagen of which the corresponding amino acid sequence is free of [Leu/Ile/Val/Met]-Xaa-Yaa-Arg wherein Xaa and Yaa correspond to Gly and Pro or other amino acids, and at least one of the amino acids between the brackets is amended could be preferred.
The process suitably provides expression leading to peptide harvest exceeding 2 g/liter or even exceeding 3 g/liter. The process can suitably be carried out with any of the recombinant collagen-like polypeptides defined above for the emulsion according to the invention. Multicopy transformants can provide more than 14 grams of gelatin per liter of clarified broth at a biomass wet weight of 435 grams per liter. Most suitably the product resulting from microbial expression is isolated and purified until it is substantially free of other protein, polysaccharides and nucleic acid. As is apparent from the examples numerous methods are available to the person skilled in the art to achieve this. The process according to the invention can provide the expression product isolated and purified to at least the following degree: content nucleic acid less than 100 ppm, content polysaccharides less than 5%, content other protein less than in commercial products. More preferably the DNA content of less than 1 ppm, RNA content less than 10 ppm even less than 5 ppm and polysaccharide content less than 0.5% or even less than 0.05% can be achieved.
The invention also concerns a process of producing an amphiphilic polymer in the manner described above, comprising introducing a gene encoding an amphiphilic polypeptide part of said polymer into a suitable host, culturing said host under conditions suitable for expression of said gene, and recovering said polypeptide. If desired the polypeptide can be coupled with another peptide or non-peptide, natural or synthetic polymer to produce a hybrid polymer suitable as an emulsifier.
In a preferred embodiment of the invention the gelatin-like material comprises no cysteine residues. The presence of cysteine in photographic product will disturb the product manufacturing process. It is thus preferred that cyteine is present in as small an amount as possible. Suitably photographic applications will employ material comprising less than 0.1% cysteine.